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T C Hall

Publications and source records attributed to T C Hall.

At least 73 records · Page 4Linked to original sources
PubMed1990

Use of Chenopodium hybridum facilitates isolation of brome mosaic virus RNA recombinants.

Three mutant brome mosaic virus (BMV) RNA-2 transcripts bearing two alterations in the pseudoknot region and one in arm C of the 3' tRNA region, previously characterized as being deficient in tRNA-like functions, have been assayed for their ability to infect and replicate (in the presence of wild-type RNAs-1 and -3) in Chenopodium hybridum plants. Although the introduced mutations have been shown to incapacitate the replication of RNA-2 in barley protoplasts, C. hybridum plants inoculated with these mutants developed local lesions indistinguishable in appearance and morphology from control inoculations containing wild-type RNA-2. Sequence analysis of progeny RNA-2 from two single lesion isolates for each mutant inoculum revealed that the input mutations were restored to functional sequences by homologous recombination within the 3'tRNA-like region. These results, which reflect the ease with which progeny RNA can be characterized from single lesions, exemplify the value of C. hybridum for studying recombination among viral RNAs.

Base Sequence↗
PubMed1990

Requirement for a viral trans-acting factor encoded by brome mosaic virus RNA-2 provides strong selection in vivo for functional recombinants.

Interaction of specific nucleotide sequences with trans-acting proteins is intrinsic to replication of viral as well as eucaryotic genomes. Brome mosaic virus RNA-2 encodes one of the two viral proteins known to be essential for replication. (R. French, M. Janda, and P. Ahlquist, Science 231:1294-1297, 1986; P. A. Kiberstis, L. S. Loesch-Fries, and T. C. Hall, Virology 112:804-808, 1981). Transfection of barley protoplasts with wild-type transcripts of brome mosaic virus RNA-1 and RNA-3 and serial dilutions of RNA-2 transcripts possessing unaltered coding sequences but bearing mutations that greatly incapacitated replication of RNA-2 revealed that trace amounts of RNA-2 are sufficient to support replication of the viral genome. In six replicate experiments containing RNA-2 transcripts devoid of the 3' 200 nucleotides that encompass the tRNA-like structure containing the minus-strand promoter, detectable levels of progeny RNA-1 and RNA-3 and subgenomic RNA-4 were present. This showed that viral p2 protein translated from the supplied RNA-2 functioned in trans to support replication of RNA-1 and RNA-3. However, in two similar experiments, progeny RNA-2 with electrophoretic mobility indistinguishable from that of wild-type RNA-2 was seen at 24 h postinoculation. Northern hybridization (RNA blot) analysis confirmed the presence of the tRNA-like 3' terminus on these progeny RNAs, indicating that recombinational restoration of the deleted sequence had occurred. This suggests that, under certain circumstances, RNA recombination may be a rapid and frequent phenomenon.

Base Sequence↗
PubMed1989

Replication in vivo of mutant brome mosaic virus RNAs defective in aminoacylation.

In order to understand the relationship between replication and aminoacylation of the genomic RNAs of brome mosaic virus, the replication of four mutants, whose RNAs were expected (on the basis of their properties in vitro) to be inefficiently tyrosylated in vivo, was studied in barley protoplasts and plants. Test inocula consisted of capped transcripts of wild-type RNAs 1 and 2, and of RNA 3 variants with defined mutations in the 3' tRNA-like region. Mutant 5'PsK, which is defective in minus-strand promoter activity and a poor substrate in vitro for both tyrosylation and 3' adenylation, replicated in protoplasts to 20% of wild-type even though only about 6% of the progeny molecules had correct 3' termini that would permit tyrosylation. Mutant psi GG, which is defective in vitro for 3' adenylation and minus-strand promoter activities but accepts tyrosine at near-normal rates, replicated to 40% of wild-type in protoplasts although only 15% of the progeny molecules had correct 3' termini. Two other mutants (delta 5' and 5'AGA), with 20-fold lower rates of tyrosylation in vitro than wild-type RNA, replicated to 60 to 70% of wild-type levels in protoplasts and gave similar yields to wild-type in systemic infections of plants. All mutant sequences were preserved in progeny RNAs, indicating that no recombination between homologous 3' ends occurred. The 40% reduction of replication in protoplasts seen for mutant delta 5', whose only known functional lesion is depressed tyrosylation in vitro, may indicate that an indirect role for aminoacylation exists. However, the results obtained argue against an obligatory role for tyrosylation in RNA replication in vivo.

Base Sequence↗
PubMed1989

Similarities among plant virus (+) and (-) RNA termini imply a common ancestry with promoters of eukaryotic tRNAs.

The 5' ends of brome mosaic virus (BMV) RNAs contain sequences similar to the consensus internal control region (ICR) of pol III promoters in tRNA genes. Comparison of BMV (+)RNA 5' termini with BMV (-)RNA termini revealed the presence of two (tandem) repeats of some 30 nucleotides, the more internal containing a region of 73% similarity to the tRNA consensus ICR2 (downstream) region of the ICR. Tandem repeats containing motifs similar to the ICR2 consensus were found at the 5' termini of (-)RNAs of cucumo-, tobamo-, and tymoviruses whose 3' (+)RNAs have aminoacylatable tRNA-like structures. Single regions of homology to the BMV(+)RNA 5' terminus, containing an ICR2-like motif, were detected for several tobravirus RNAs, and for satellite tobacco necrosis virus RNA. The (+)-stranded genomes of these viruses have not been shown to be capable of amino acid esterification. The ICR2 consensus (GGUUCGANUCC) is nearly palindromic, and is contained with the T psi C loop of tRNAs and viral analogs. Consequently, tRNA promoter-like motifs can be seen at both termini of (+) and (-) RNAs of bromoviruses and other viruses. The presence of ICR1 and ICR2-like sequences in BMV genomic 5' (+)RNAs and the tobamovirus 5' (-)RNAs may reflect promoter arrangements of primordial genomic RNAs ancestral to both modern plant viruses and eukaryotic tRNAs. Several derivative concepts related to genome evolution are discussed, including the origin of asymmetric strand synthesis of RNAs.

Base Sequence↗
PubMed1989

Imaging of colorectal carcinoma with radiolabeled antibodies.

Colorectal cancer has been the tumor type most frequently studied with radiolabeled antibodies. Among the various antibodies, a majority of patients with colorectal cancer have received xenogeneic polyclonal or monoclonal antibodies against carcino-embryonic antigen. This review summarizes the current status of colorectal cancer imaging with radiolabeled antibodies, ie, radioimmunodetection (RAID), and examines the published studies involving carcinoembryonic antigen (CEA) antibodies and 17-1A, 19-9, and B72.3, and other monoclonal antibodies. In order to better address the issue of the current and future clinical usefulness of this emerging technology, particular attention is given to the protocols, methods, and results of the published studies. Despite differences in study parameters, antibodies and forms, labels, administration routes and doses, and scanning instruments and methods, it has been found that (1) almost no adverse reactions have been evident; (2) antibody fragments are preferred over whole immunoglobulin G reagents because they achieve higher tumor-to-background ratios earlier, thus reducing or precluding the need for dual-isotope subtraction methods or long delays before imaging; (3) use of antibody fragments, including the monovalent Fab' form, permits imaging with short-lived radionuclides of excellent photon properties, such as 123I and 99mTc; (4) circulating antigens against which the imaging antibody is directed can complex with the injected antibody, but such complexes have not prevented successful RAID; (5) patients with high serum titers of the appropriate antigen target usually have higher rates of positive RAID; (6) patients who are seronegative for the tumor antigen being studied can have positive RAID findings, which can represent the detection of occult lesions; (7) single photon emission computed tomography appears to provide better image resolution than planar scanning; (8) regardless of the sensitivity reported in any particular study, almost all investigators have observed the disclosure of occult neoplasms by RAID; and (9) RAID, a more functional test of usually high specificity, can complement other radiological methods, such as computed tomography scans, which are limited to structural information.

Antibodies, Monoclonal↗
PubMed1989

Telomeric function of the tRNA-like structure of brome mosaic virus RNA.

Four mutant brome mosaic virus (BMV) RNA3 transcripts, bearing single or double base changes in the 3'-CCAOH terminus of the tRNA-like structure, previously characterized as being deficient in vitro with respect to aminoacylation and replication activities, have now been assayed in vivo for their ability to replicate (in the presence of transcripts of wild-type RNA1 and -2) in barley protoplasts and plants. In tests conducted with protoplasts, irrespective of the time post-infection, all four mutants were fully viable, and the relative levels of both plus and minus strand replication for each mutant were similar to that of the wild type. Inoculation of barley plants with these mutants resulted in phenotypic symptoms and viral yields that were similar to those from wild-type infections. Analysis of each mutant progeny RNA3 indicated that the altered sequence at the 3' terminus was restored to that of wild type. These observations indicate that there is a rapid turnover and correction of the 3' termini of BMV RNAs in vivo. Such correction is commensurate with the action of tRNA nucleotidyltransferase, but it differs from recombination processes that appear to be relatively infrequent for BMV RNA3. These results support the conclusion that the 3'-CCAOH termini of viral tRNA-like structures function analogously to telomeres of chromosomal DNA.

Base Sequence↗
PubMed1989

Regulation of beta-glucuronidase expression in transgenic tobacco plants by an A/T-rich, cis-acting sequence found upstream of a French bean beta-phaseolin gene.

A 0.8-kilobase fragment from the 5'-flanking region of a French bean beta-phaseolin gene yielded strong, temporally regulated, and embryo-specific expression of beta-glucuronidase (GUS) in transgenic tobacco plants, paralleling that found for the seed protein phaseolin [Sengupta-Gopalan, C., Reichert, N.A., Barker, R.F., Hall. T.C., and Kemp, J.D. (1985) Proc. Natl. Acad. Sci. USA 82, 3320-3324]. Gel retardation and footprinting assays using nuclear extracts from immature bean cotyledons revealed strong binding of nuclear proteins to an upstream region (-628 to -682) that contains two inverted A/T-rich motifs. Fusion of a 103-base pair fragment or a 55-base pair synthetic oligonucleotide containing these motifs to a minimal 35S promoter/GUS cassette yielded strong GUS expression in several tissues. A different pattern of GUS expression was obtained in immature embryos and germinating seedlings from the nominally constitutive, full-length, 35S promoter. Whereas GUS expression under the control of the 0.8-kilobase beta-phaseolin regulatory region is limited to immature embryos, expression from constructs containing the A/T-rich motifs is strongest in roots. These data, combined with S1 mapping, provide direct evidence that a plant upstream A/T-rich sequence that binds nuclear proteins can activate transcription in vivo. They also indicate that additional regulatory elements in the beta-phaseolin 5'-flanking region are required for embryo-specific gene expression.

Base Composition↗
PubMed1988

Mutational analysis of the sequence and structural requirements in brome mosaic virus RNA for minus strand promoter activity.

An RNA-dependent RNA polymerase (replicase) activity that specifically copies brome mosaic virus (BMV) RNAs in vitro can be prepared from BMV-infected barley leaves. The signals directing complementary (minus) strand synthesis reside within the 3' 134-nucleotide-long tRNA-like structure that is common to each of the virion RNAs. By studying the influence of minus strand synthesis of numerous mutations introduced throughout this region of the RNA, we have mapped in detail the sequence and structural elements necessary for minus strand promoter activity. Sequence alterations (either substitutions or small, structurally discrete deletions) in most parts of the tRNA-like structure resulted in decreased minus strand synthesis. This suggests that BMV replicase is a large enzyme, possibly composed of several subunits. The lowest activities, 5 to 8% of wild type, were observed for mutants with substitutions at three separate loci, identifying one structural and two sequence-specific elements essential for optimal promoter activity. (1) Destabilization of the pseudoknot structure in the aminoacyl acceptor stem resulted in low promoter activity, demonstrating the importance of a tRNA-like conformation. (2) Substitution of the C residue adjacent to the 3' terminus resulted in low promoter activity, probably by interfering with strand initiation. (3) The low activities resulting from substitutions and a small deletion in arm C suggest this region of the RNA to be a major feature involved in replicase binding. In particular, nucleotides within the loop of arm C appear to be involved in a sequence-specific interaction with the replicase.

Autoradiography↗
PubMed1988

Mutational analysis of the tRNA mimicry of brome mosaic virus RNA. Sequence and structural requirements for aminoacylation and 3'-adenylation.

The genomic RNAs of brome mosaic virus (BMV) exhibit various tRNA-like properties, including specific tyrosylation by tyrosyl-tRNA synthetases and adenylation of the 3'-CCOH derivative by tRNA nucleotidyl transferases. We have studied the effect of numerous mutations in all domains of the tRNA-like structure of BMV RNA on tyrosylation and adenylation in vitro. Surprisingly few mutations resulted in more than 50% decrease in tyrosylation rates with either wheat germ or yeast synthetases; those mutations were at the 3' terminus, the pseudoknot, and the bases of arms B and E. The results suggest an interaction of synthetase with arm A as the analog of the aminoacyl acceptor stem of tRNAs, and arm B as the analog of the anticodon arm of tRNAs, although there is no apparent interaction with the terminal loop of arm B analogous to the interaction with the anticodon in tRNAs. Mutations at several loci resulted in large losses of adenylation activity catalyzed by wheat germ and Escherichia coli nucleotidyl transferases; those loci were the pseudoknot, the bases of arms B, C and D, and at the junctions of these arms with arm A. These studies have identified mutants specifically defective in one of the tRNA-like activities, which are appropriate for investigating the role of these activities during infection in vivo.

Autoradiography↗
PubMed1988

Mutational analysis of the core and modulator sequences of the BMV RNA3 subgenomic promoter.

The subgenomic promoter of a (+)-stranded RNA virus, brome mosaic virus (BMV) controlling synthesis of subgenomic RNA4 has been defined in vitro. Truncated and mutant (-)-strand RNA templates were produced by in vitro transcription of cloned RNA3 cDNA. Subgenomic (+)-sense RNA was synthesized in vitro from these templates by a replicase (RNA-dependent RNA polymerase) preparation extracted from infected barley leaves. The activities of templates with truncations and deletions surrounding the RNA4 initiation site revealed a promoter of approximately 62 bases grouped into four functional domains. The core sequence consists of about twenty bases immediately upstream of, and including, the initiation nucleotide. In addition to the core sequence, a domain overlapping the 5' untranslated end of RNA4 apparently determines correct initiation. Two domains immediately upstream of the promoter core consist of the internal poly(A) tract of RNA3, which probably serves as an non base-paired spacer facilitating access of the replicase to the promoter, and a sequence, UUAUUAUU, that is required for high levels of promoter activity. Homologies to sequences surrounding the initiation sites of subgenomic RNAs from several plant RNA viruses, and from alphaviruses, have been detected.

Alphavirus↗
PubMed1987

Analysis of brome mosaic virus replication and aminoacylation functions by site-specific mutagenesis.

Brome Mosaic Virus (BMV) has a tripartite RNA genome; each RNA and the subgenomic RNA encoding the viral coat protein share a highly homologous region of about 200 nucleotides at the 3' end, for which a tRNA-like structure has been proposed. Several sequences encoding functions, including replicase binding, initiation of (-) strand synthesis and tyrosine esterification are known to be nested within this region. Elongation factor EF-1 alpha binds to aminoacylated viral RNAs, but not to the uncharged forms. An additional function of the tRNA-like structure is to serve as a substrate for nucleotidyl transferase, which adds the terminal adenosine residue to the (+) sense virion RNAs. A template-dependent and template-specific replicase preparation from BMV-infected barley leaves has been characterized and extensively used for replication studies in vitro that complement studies in vivo using protoplasts. The replicase has been shown to initiate de novo both (-) strand synthesis on supplied (+) strand RNAs, and (+) strand subgenomic RNA synthesis on supplied (-) sense RNA3 templates. RNA transcripts obtained by transcription in vitro of cDNA clones containing desired base substitutions and deletions, have been supplied as templates for replication, aminoacylation and other assays. Use of such mutant RNAs has allowed the promoters for both (-) strand synthesis and for synthesis of the subgenomic (+) strand RNA to be characterized and defined. The same approach has also been used to reveal regions of the tRNA-like structure involved in the tyrosylation of the BMV RNAs. These experiments showed that, although regions important in aminoacylation and replication functions overlap, they are not identical. Some of the mutations tested in vitro have also been tested for infectivity in vivo using both barley plants and protoplasts. Mutants retaining replicase and nucleotidyl transferase template activity, but having lost aminoacylation capability are of special interest in that they should reveal the role of aminoacylation in the infection process.

Base Sequence↗
PubMed1986

Minus-strand initiation by brome mosaic virus replicase within the 3' tRNA-like structure of native and modified RNA templates.

An RNA-dependent RNA polymerase (replicase) extract from brome mosaic virus-infected barley leaves has been shown to initiate synthesis of (-) sense RNA from (+) sense virion RNA. Initiation occurred de novo, as demonstrated by the incorporation of [gamma-32P]GTP into the product. Sequencing using cordycepin triphosphate to terminate (-) strands during their synthesis by the replicase generated sequence ladders that confirmed that copying was accurate, and that initiation occurred very close to the 3' end. The precise site of initiation was further defined by testing the replicase template activity after stepwise removal of 3'-terminal nucleotides. Whereas removal of the terminal A did not decrease template activity, removal of the next nucleotide (C-2) did. Thus, initiation almost certainly occurs opposite the penultimate 3'-nucleotide (C-2) in vitro. The structure of the double-stranded replicative form of RNA isolated from brome mosaic virus-infected leaves was consistent with such a mechanism occurring in vivo, in that it lacked the 3'-terminal A found on virion RNAs. The specific site of (-) strand initiation and normal template activity were retained for RNAs with as many as 15 to 30 A residues added to the 3' end. However, only limited oligonucleotide 3' extensions can be present on active templates. In order to assess the 5' extent of sequences required for an active template, a 134-nucleotide-long fragment of brome mosaic virus RNA, corresponding to the tRNA-like structure, was generated. This RNA had high template activity, but a shorter 3' (85-nucleotide) fragment was inactive. RNAs with various heterologous sequences 5' to position 134 also showed high template activity. Thus, the 3'-terminal tRNA-like structure common to all four brome mosaic virus virion RNAs contains all of the signals required for initiation of replication, and sequences 5' to it do not play a role in template selection.

Base Sequence↗
PubMed1986

Modulation of replication, aminoacylation and adenylation in vitro and infectivity in vivo of BMV RNAs containing deletions within the multifunctional 3' end.

The genome of brome mosaic virus (BMV) is comprised of three (+) strand RNAs, each containing a similar, highly structured, 200 base long sequence at its 3' end. A 134 base subset of this sequence contains signals directing interaction of the viral RNA with BMV RNA replicase, ATP,CTP:tRNA nucleotidyl transferase and aminoacyl tRNA synthetase. A series of mutants containing deletions within this region, previously constructed and tested in vitro for the effect on replication and aminoacylation activities, has now been assayed in vitro for adenylation function and in vivo for ability to replicate in isolated protoplasts and whole plants. These tests indicate that features of viral RNA recognized by BMV replicase overlap those directing adenylation, but are distinct from those directing aminoacylation. Consequently, the lethality of a deletion preferentially inhibiting aminoacylation suggests that this function may have an essential role contributing to viral replication in vivo. An RNA3 mutant bearing a 20-base deletion yielding normal levels of aminoacylation and enhanced levels of replicase template activity and adenylation in vitro was able to replicate in protoplasts and plants; however, its accumulation in protoplasts was reduced relative to wild-type. This suggests that additional functions affecting the replication and accumulation of viral RNA reside in the conserved 3' sequence.

Base Sequence↗